Analyzing Biological and Organic Polymers by MALDI-TOF Jonathan A. Karty, Ph.D.
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Transcript of Analyzing Biological and Organic Polymers by MALDI-TOF Jonathan A. Karty, Ph.D.
Analyzing Biological and Analyzing Biological and Organic Polymers by Organic Polymers by
MALDI-TOFMALDI-TOFJonathan A. Karty, Ph.D.Jonathan A. Karty, Ph.D.
Topics CoveredTopics Covered
Sample RequirementsSample Requirements
Instrument OverviewInstrument Overview
General Instrument Use InstructionsGeneral Instrument Use Instructions
Tips and TricksTips and Tricks
What is the Bruker Autoflex III?What is the Bruker Autoflex III? Time-of-flight mass spectrometerTime-of-flight mass spectrometer
Ions of given same kinetic energy, heavy ions travel Ions of given same kinetic energy, heavy ions travel slower than lighter onesslower than lighter ones
Two modes of operationTwo modes of operation LinearLinear ReflectronReflectron
MALDI/LDI sourceMALDI/LDI source 384 position target plate (~1 384 position target plate (~1 µµL spot size)L spot size) 355 nm Nd:YAG laser355 nm Nd:YAG laser Can analyze positive or negative ions (same spot)Can analyze positive or negative ions (same spot)
Matrix-Assisted Laser Desorption/Ionization Matrix-Assisted Laser Desorption/Ionization (MALDI)(MALDI)
Analyte is mixed with UV-absorbing matrixAnalyte is mixed with UV-absorbing matrix ~10,000:1 matrix:analyte ratio~10,000:1 matrix:analyte ratio Analyte does not need to absorb laserAnalyte does not need to absorb laser
A drop of this liquid is dried on a targetA drop of this liquid is dried on a target Analyte incorporated into matrix crystalsAnalyte incorporated into matrix crystals
Spot is irradiated by a laser pulseSpot is irradiated by a laser pulse Irradiated region sublimes, taking analyte with itIrradiated region sublimes, taking analyte with it Matrix is often promoted to the excited stateMatrix is often promoted to the excited state Charges exchange between matrix and analyte in the Charges exchange between matrix and analyte in the
plume (very fast <100 nsec)plume (very fast <100 nsec)
Ions are accelerated toward the detectorIons are accelerated toward the detector
MALDI AdvantagesMALDI Advantages Technique is relatively simpleTechnique is relatively simple Volatilize and ionize labile molecules Volatilize and ionize labile molecules
Imagine electron ionization on a proteinImagine electron ionization on a protein MALDI creates very simple mass spectraMALDI creates very simple mass spectra
Ions are usually (M+nH)Ions are usually (M+nH)n+n+ or (M-nH) or (M-nH)n-n-
Only 1-3 charge states are observedOnly 1-3 charge states are observed Usually 1 charge state for peptides < 3.5 kDaUsually 1 charge state for peptides < 3.5 kDa
MALDI ideal for time-of-flight analyzersMALDI ideal for time-of-flight analyzers Theoretically unlimited mass range (100 kDa done here)Theoretically unlimited mass range (100 kDa done here)
MALDI is very rapid (<1 min/spot)MALDI is very rapid (<1 min/spot) Low sample consumption (1 Low sample consumption (1 µµL)L) Wide array of matrices available for different analytesWide array of matrices available for different analytes
Some Common MALDI MatricesSome Common MALDI Matrices
C
N
C
O
OH
HO O
O
HO
H3C
CH3
C
O
OH
C
O
OH
OH
HO
NH
C
O
OHC
N
C
N
C
N
C
N
-cyano-4-hydroxycinnamic acidCCA C10H7NO3
peptides and small molecules
sinapinic acidSA C11H12O5
proteins
2,5 dihydroxybenzoic acidDHB C7H6O4
oligosaccharides
N
3-indoleacrylic acidIAA C10H9NO2
organic polymers
7,7,8,8 tetracyanoquinodimethaneTCNQ C12H4N4
polyaromatic hydrocarbons
C
O
OH
OH
3-hydroxypicolinic acidHPA C6H5NO3oligonucleotides
What Samples Can It Run?What Samples Can It Run? BiopolymersBiopolymers
Peptides, proteins, DNA, RNA, oligosaccharidesPeptides, proteins, DNA, RNA, oligosaccharides Organometallic complexesOrganometallic complexes
Organometallic salts work greatOrganometallic salts work great Synthetic polymersSynthetic polymers
Polymer need not be soluble in same solvent as Polymer need not be soluble in same solvent as matrixmatrix
Molecules that photoionize upon irradiation by Molecules that photoionize upon irradiation by 355 nm laser355 nm laser PorphyrinsPorphyrins Organometallic complexesOrganometallic complexes
What Samples Can’t It Run?What Samples Can’t It Run? ““Dirty” samplesDirty” samples
Significant concentration of involatilesSignificant concentration of involatiles Glycerol, urea, most buffers, many detergentsGlycerol, urea, most buffers, many detergents
Alkali metal salts can be quite problematicAlkali metal salts can be quite problematic RNA/DNA analyses require extensive desaltingRNA/DNA analyses require extensive desalting
Molecules with significant vapor pressuresMolecules with significant vapor pressures Instrument is held at ~10Instrument is held at ~10-7-7 torr torr
Molecules that do not make stable ions in Molecules that do not make stable ions in sourcesource Lack charge acceptor/donor siteLack charge acceptor/donor site Cannot photoionize with Nd:YAG laserCannot photoionize with Nd:YAG laser
MALDI AdvantagesMALDI Advantages
Relatively gentle ionization techniqueRelatively gentle ionization techniqueVery high MW species can be ionizedVery high MW species can be ionizedMolecule need not be volatileMolecule need not be volatileVery easy to get sub-picomole sensitivityVery easy to get sub-picomole sensitivitySpectra are easy to interpretSpectra are easy to interpretPositive or negative ions from same spotPositive or negative ions from same spotWide array of matrices availableWide array of matrices available
MALDI DisadvantagesMALDI DisadvantagesMALDI matrix cluster ions can obscure low MALDI matrix cluster ions can obscure low
m/z (<600) rangem/z (<600) rangeAnalyte must have very low vapor pressureAnalyte must have very low vapor pressureCoupling MALDI with chromatography can Coupling MALDI with chromatography can
be difficultbe difficultAnalytes that absorb the laser can be Analytes that absorb the laser can be
problematicproblematicFluorescein-labeled peptidesFluorescein-labeled peptides
Instrument DiagramInstrument Diagram
ReflectorDetector
LinearDetectorLens
Target
ExtractionPlate
FlightTube
Entrance
Reflectron
355 nm Nd:YAG laser
Linear ModeLinear Mode
ReflectorDetector
LinearDetectorLens
Target
ExtractionPlate
FlightTube
Entrance
Reflectron
355 nm Nd:YAG laser
Linear mode is used for large (>3.5 kDa) molecules or
exceedingly fragile species (oligosaccharides). It is capable of 4,000 resolving power @ 3.2
kDa (1,000 RP @ 12 kDa)
Reflectron ModeReflectron Mode
ReflectorDetector
LinearDetectorLens
Target
ExtractionPlate
FlightTube
Entrance
Reflectron
355 nm Nd:YAG laser
Reflectron mode is used for small species (<4 kDa) and is capable of 11,000 resolving
power @ 3.2 kDa.
MALDI ExampleMALDI Example
(Ubiq+H)+
(Ins+H)+(Ub
iq+
2H
)2+
(ACTH 7-38+H)+(A
CT
H 1
8-3
7+
H)+
O
N
N
O
HN
HN
O
O
C12H25O
C12H25O
C12H25O
C12H25O
OC12H25
OC12H25
O O OO
N
N
O
NH
NH
O
O
OC12H25
OC12H25
OC12H25
OC12H25
C12H25O
C12H25O
Chemical Formula: C230H354N8O23
Exact Mass: 3596.68
8476.61
16952.53
4802.98
0
2000
4000
6000
Inte
ns.
[a.u
.]
5000 7500 10000 12500 15000 17500 20000 22500 25000 27500m/z
16952.53
17131.5417056.51
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
16600 16800 17000 17200 17400 17600m/z
MALDI-TOF Example 2MALDI-TOF Example 2
General Sample GuidelinesGeneral Sample Guidelines Purify analyte if possiblePurify analyte if possible
Analyte should be 5 – 100 Analyte should be 5 – 100 µµM in concentrationM in concentration ZipTips can help purify dirty samples (CZipTips can help purify dirty samples (C44 and C and C1818
available in MSF)available in MSF) Use only volatile solvents/buffersUse only volatile solvents/buffers
MeOH, HMeOH, H22O, acetone, CHO, acetone, CH33CN, THF, CHCN, THF, CH22ClCl22, C, C66HH66
TFA, HOAc, formic acid, NHTFA, HOAc, formic acid, NH33, etc., etc. Ionic strength < 20 mM (e.g. 0.1% v/v HOAc)Ionic strength < 20 mM (e.g. 0.1% v/v HOAc)
If you need a detergent, 20 mM n-If you need a detergent, 20 mM n-octylglucopyranoside can workoctylglucopyranoside can work No SDS, TWEEN, CHAPS, etcNo SDS, TWEEN, CHAPS, etc
Need at least 2 Need at least 2 µµLL
Sample Prep TricksSample Prep Tricks Ziptip to clean up dirty samplesZiptip to clean up dirty samples
CC1818 for peptides < 3 kDa for peptides < 3 kDa CC44 for peptides/proteins > 3kDa for peptides/proteins > 3kDa Elute directly into matrix for added sensitivityElute directly into matrix for added sensitivity ZipTip instructions on MSF websiteZipTip instructions on MSF website
If CCA liquid turns yellow, pH is too highIf CCA liquid turns yellow, pH is too high Spots from non-acidic CCA do not crystallize correctlySpots from non-acidic CCA do not crystallize correctly Add a little 1% v/v or 10% v/v TFA to lower pHAdd a little 1% v/v or 10% v/v TFA to lower pH If sample needs base for solubility, try over-layer If sample needs base for solubility, try over-layer
methodmethod Dissolve sample in NHDissolve sample in NH33 or other volatile base or other volatile base Place 1 uL of sample on target, let dry completelyPlace 1 uL of sample on target, let dry completely Deposit 1 uL matrix over top of dried sampleDeposit 1 uL matrix over top of dried sample
Sample Prep Tricks 2Sample Prep Tricks 2
Non-aqueous over-layerNon-aqueous over-layerMake 1 uL spot of matrix on plate, let dryMake 1 uL spot of matrix on plate, let dryDeposit small amount of sample in volatile Deposit small amount of sample in volatile
solvent (e.g. CHClsolvent (e.g. CHCl33, acetone, CH, acetone, CH22ClCl22))You can even do internal calibration this wayYou can even do internal calibration this way
Put calibrants in matrix spotPut calibrants in matrix spot
For better mass accuracy, let voltages For better mass accuracy, let voltages stabilize 10-30 minutes before recording stabilize 10-30 minutes before recording datadata
Hands-on TrainingHands-on Training
Starts AFTER 11/7Starts AFTER 11/7Groups of no more than threeGroups of no more than threeOne hour or so to completeOne hour or so to completeNo charge for first sessionNo charge for first sessionAfter training, students must demonstrate After training, students must demonstrate
competency by running their own samples competency by running their own samples prior to being granted after-hours accessprior to being granted after-hours access